The ultimate goal of the research described in this application is to develop an economical, safe and effective therapeutic that can reverse the paralytic symptoms of botulism. Success in achieving this goal could save countless lives in the event of a bioterrorist attack by botulinum toxin. The goal of administering an anti-toxin antibody is rapid and complete removal of the toxin from the body. Currently available immunotherapies for botulism (equine antitoxin, human botulism immune globulin, monoclonal antibodies) can remove only toxin molecules that have not yet reached motor neurons. Once the catalytic light chain of the toxin reaches the cytosol of motor neurons, where it cleaves proteins involved in neurotransmission, it is inaccessible to normal antibodies. This is why victims of botulinum neurotoxin poisoning may need supportive care on a respirator for many weeks. We propose using a new single-chain variable fragment, a nanobody, against the catalytic site of the toxin light chain, along with technology that allows proteins to penetrate into the cytosol of motor neurons, to reverse the paralysis caused by the toxin. We will start with a single-domain variable fragment, isolated from a llama antibody library in the laboratory of our collaborator, Dr James Marks (UCSF), that inhibits the ability of the toxin light chain to cleave it's protein substrate. We will express fusions of this nanobody to various cell-penetrating peptides, using a plant expression system. We will then test the ability of these nanobody fusions to enter primary rat neuron cells and inhibit toxin activity. Finally, we will test the toxicity of the cell-penetrating nanobody in mice. PUBLIC HEALTH RELEVANCE: Botulinum toxin is the deadliest biological substance known, and would be easy for a terrorist to produce and use to cause large numbers of casualties. This makes developing an effective and economical countermeasure to protect the public a public health priority. The research supported by this grant will lay the groundwork for using plants to produce a highly protective, safe and economical antitoxin therapy.